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1.
Am J Physiol Heart Circ Physiol ; 310(10): H1313-20, 2016 05 15.
Article in English | MEDLINE | ID: mdl-26993228

ABSTRACT

Late Na(+) current (INaL) is enhanced in myocytes of animals with chronic heart failure and patients with hypertrophic cardiomyopathy. To define the role of INaL in diastolic heart failure, the effects of GS-458967 (GS-967), a potent INaL inhibitor on mechanical and electrical abnormalities, were determined in an animal model of diastolic dysfunction. Dahl salt-sensitive (DSS) rats fed a high-salt (HS) diet for 8 wk, compared with a normal salt (NS) diet, had increased left ventricular (LV) mass (1,257 ± 96 vs. 891 ± 34 mg) and diastolic dysfunction [isovolumic relaxation time (IVRT): 26.8 ± 0.5 vs. 18.9 ± 0.2 ms; early transmitral flow velocity/early mitral annulus velocity (E/E') ratio: 25.5 ± 1.9 vs. 14.9 ± 0.9]. INaL in LV myocytes from HS rats was significantly increased to 0.41 ± 0.02 from 0.14 ± 0.02 pA/pF in NS rats. The action potential duration (APD) was prolonged to 136 ± 12 from 68 ± 9 ms in NS rats. QTc intervals were longer in HS vs. NS rats (267 ± 8 vs. 212 ± 2 ms). Acute and chronic treatment with GS-967 decreased the enhanced INaL to 0.24 ± 0.01 and 0.17 ± 0.02 pA/pF, respectively, vs. 0.41 ± 0.02 pA/pF in the HS group. Chronic treatment with GS-967 dose-dependently reduced LV mass, the increases in E/E' ratio, and the prolongation of IVRT by 27, 27, and 20%, respectively, at the 1.0 mg·kg(-1)·day(-1) dose without affecting blood pressure or LV systolic function. The prolonged APDs in myocytes and QTc of HS rats were significantly reduced with GS-967 treatment. These results indicate that INaL is a significant contributor to the LV diastolic dysfunction, hypertrophy, and repolarization abnormalities and thus, inhibition of this current is a promising therapeutic target for diastolic heart failure.


Subject(s)
Anti-Arrhythmia Agents/pharmacology , Arrhythmias, Cardiac/drug therapy , Heart Conduction System/drug effects , Heart Failure/drug therapy , Myocytes, Cardiac/drug effects , Pyridines/pharmacology , Sodium Channel Blockers/pharmacology , Sodium Channels/drug effects , Triazoles/pharmacology , Ventricular Dysfunction, Left/drug therapy , Ventricular Function, Left/drug effects , Action Potentials/drug effects , Animals , Arrhythmias, Cardiac/metabolism , Arrhythmias, Cardiac/physiopathology , Disease Models, Animal , Dose-Response Relationship, Drug , Heart Conduction System/metabolism , Heart Conduction System/physiopathology , Heart Failure/metabolism , Heart Failure/physiopathology , Heart Rate/drug effects , Hypertrophy, Left Ventricular/metabolism , Hypertrophy, Left Ventricular/physiopathology , Hypertrophy, Left Ventricular/prevention & control , Male , Myocardial Contraction/drug effects , Myocytes, Cardiac/metabolism , Oxidative Stress/drug effects , Rats, Inbred Dahl , Sodium Channels/metabolism , Sodium Chloride, Dietary , Time Factors , Ventricular Dysfunction, Left/metabolism , Ventricular Dysfunction, Left/physiopathology
2.
Am J Physiol Heart Circ Physiol ; 310(3): H426-35, 2016 Feb 01.
Article in English | MEDLINE | ID: mdl-26637557

ABSTRACT

Pathological enhancement of late Na(+) current (INa) can potentially modify intracellular ion homeostasis and contribute to cardiac dysfunction. We tested the hypothesis that modulation of late INa can be a source of intracellular Na(+) ([Na(+)]i) overload. Late INa was enhanced by exposing rabbit ventricular myocytes to Anemonia sulcata toxin II (ATX-II) and measured using whole cell patch-clamp technique. [Na(+)]i was determined with fluorescent dye Asante NaTRIUM Green-2 AM. Pacing-induced changes in the dye fluorescence measured at 37°C were more pronounced in ATX-II-treated cells than in control (dye washout prevented calibration). At 22-24°C, resting [Na(+)]i was 6.6 ± 0.8 mM. Treatment with 5 nM ATX-II increased late INa 8.7-fold. [Na(+)]i measured after 2 min of electrical stimulation (1 Hz) was 10.8 ± 1.5 mM and 22.1 ± 1.6 mM (P < 0.001) in the absence and presence of 5 nM ATX-II, respectively. Inhibition of late INa with GS-967 (1 µM) prevented Na(+) i accumulation. A strong positive correlation was observed between the late INa and the pacing-induced increase of [Na(+)]i (R(2) = 0.88) and between the rise in [Na(+)]i and the increases in cytosolic Ca(2+) (R(2) = 0.96). ATX-II, tetrodotoxin, or GS-967 did not affect [Na(+)]i in quiescent myocytes suggesting that late INa was solely responsible for triggering the ATX-II effect on [Na(+)]i. Experiments with pinacidil and E4031 indicate that prolongation of the action potential contributes to as much as 50% of the [Na(+)]i overload associated with the increase in late INa caused by ATX-II. Enhancement of late INa can cause intracellular Na(+) overload in ventricular myocytes.


Subject(s)
Calcium/metabolism , Cardiotonic Agents/pharmacology , Cnidarian Venoms/pharmacology , Myocytes, Cardiac/drug effects , Sodium Channels/drug effects , Sodium/metabolism , Animals , Green Fluorescent Proteins , Heart Ventricles/cytology , Indoles , Myocytes, Cardiac/metabolism , Optical Imaging , Patch-Clamp Techniques , Rabbits , Sodium Channels/metabolism
3.
Bioorg Med Chem Lett ; 26(13): 3207-3211, 2016 07 01.
Article in English | MEDLINE | ID: mdl-27038498

ABSTRACT

Previously we disclosed the discovery of potent Late INa current inhibitor 2 (GS-458967, IC50 of 333nM) that has a good separation of late versus peak Nav1.5 current, but did not have a favorable CNS safety window due to high brain penetration (3-fold higher partitioning into brain vs plasma) coupled with potent inhibition of brain sodium channel isoforms (Nav1.1, 1.2, 1.3). We increased the polar surface area from 50 to 84Å(2) by adding a carbonyl to the core and an oxadiazole ring resulting in 3 GS-462808 that had lower brain penetration and serendipitously lower activity at the brain isoforms. Compound 3 has an improved CNS window (>20 rat and dog) relative to 2, and improved anti-ischemic potency relative to ranolazine. The development of 3 was not pursued due to liver lesions in 7day rat toxicology studies.


Subject(s)
Azoles/pharmacology , Drug Discovery , Heart/drug effects , NAV1.5 Voltage-Gated Sodium Channel/metabolism , Pyridines/pharmacology , Ranolazine/pharmacology , Sodium Channel Blockers/pharmacology , Animals , Azoles/chemical synthesis , Azoles/chemistry , Dogs , Dose-Response Relationship, Drug , Ether-A-Go-Go Potassium Channels/antagonists & inhibitors , Ether-A-Go-Go Potassium Channels/metabolism , Haplorhini , Humans , Molecular Structure , Pyridines/chemical synthesis , Pyridines/chemistry , Rabbits , Ranolazine/chemical synthesis , Ranolazine/chemistry , Rats , Sodium Channel Blockers/chemical synthesis , Sodium Channel Blockers/chemistry , Structure-Activity Relationship
4.
Mol Pharmacol ; 85(1): 162-74, 2014 Jan.
Article in English | MEDLINE | ID: mdl-24202911

ABSTRACT

Ranolazine is an approved drug for chronic stable angina that acts by suppressing a noninactivating current conducted by the cardiac sodium channel [persistent sodium ion current (INa)]. Ranolazine has also been shown to inhibit the increased persistent INa carried by NaV1.1 channels encoding epilepsy- and migraine-associated mutations. Here, we investigate the antiepileptic properties of ranolazine exhibited through the reduction of hippocampal neuronal excitability. At therapeutically relevant concentrations, ranolazine reduced action potential firing frequency of hippocampal neurons in response to repetitive depolarizing current injections. Similarly, using a single current injection paradigm, ranolazine required a long depolarization (4 seconds) to produce significant inhibition of excitability, which was similar to that observed for the anticonvulsants phenytoin (slowly binds to the fast-inactivated state) and lacosamide (binds to the slow-inactivated state). Ranolazine enhanced the development of fast and slow inactivation assessed with conditioning prepulses of 100, 1000, or 10,000 milliseconds. Recovery of channels from inactivated states was also slowed in the presence of ranolazine. Interestingly, the use-dependent inhibition of hippocampal neurons was dependent on the duration of the voltage step, suggesting ranolazine does not selectively affect the open state and may also interact with inactivated states. NEURON (Yale University, New Haven, CT) computational simulations predict equal inhibition of action potential generation for binding to either fast-inactivated or slow-inactivated states. Binding of ranolazine to either preopen or open states did not affect the excitability of the simulation. Ranolazine was able to significantly reduce the epileptiform activity of the neuronal cultures, suggesting possible antiepileptic activity.


Subject(s)
Acetanilides/pharmacology , Anticonvulsants/pharmacology , Hippocampus/drug effects , Neurons/drug effects , Piperazines/pharmacology , Voltage-Gated Sodium Channels/physiology , Action Potentials/drug effects , Animals , Cells, Cultured , Computer Simulation , Epilepsy/physiopathology , Hippocampus/cytology , Hippocampus/metabolism , Humans , Markov Chains , N-Methylaspartate/pharmacology , NAV1.1 Voltage-Gated Sodium Channel/chemistry , NAV1.1 Voltage-Gated Sodium Channel/physiology , NAV1.2 Voltage-Gated Sodium Channel/chemistry , NAV1.2 Voltage-Gated Sodium Channel/physiology , Neurons/physiology , Patch-Clamp Techniques , Protein Binding , Protein Conformation , Ranolazine , Rats , Voltage-Gated Sodium Channels/chemistry
5.
J Pharmacol Exp Ther ; 350(2): 455-68, 2014 Aug.
Article in English | MEDLINE | ID: mdl-24917542

ABSTRACT

Both preclinical evidence and clinical evidence suggest that α7 nicotinic acetylcholine receptor activation (α7nAChR) improves cognitive function, the decline of which is associated with conditions such as Alzheimer's disease and schizophrenia. Moreover, allosteric modulation of α7nAChR is an emerging therapeutic strategy in an attempt to avoid the rapid desensitization properties associated with the α7nAChR after orthosteric activation. We used a calcium assay to screen for positive allosteric modulators (PAMs) of α7nAChR and report on the pharmacologic characterization of the novel compound RO5126946 (5-chloro-N-[(1S,3R)-2,2-dimethyl-3-(4-sulfamoyl-phenyl)-cyclopropyl]-2-methoxy-benzamide), which allosterically modulates α7nAChR activity. RO5126946 increased acetylcholine-evoked peak current and delayed current decay but did not affect the recovery of α7nAChRs from desensitization. In addition, RO5126946's effects were absent when nicotine-evoked currents were completely blocked by coapplication of the α7nAChR-selective antagonist methyl-lycaconitine. RO5126946 enhanced α7nAChR synaptic transmission and positively modulated GABAergic responses. The absence of RO5126946 effects at human α4ß2nAChR and 5-hydroxytryptamine 3 receptors, among others, indicated selectivity for α7nAChRs. In vivo, RO5126946 is orally bioavailable and brain-penetrant and improves associative learning in a scopolamine-induced deficit model of fear conditioning in rats. In addition, procognitive effects of RO5126946 were investigated in the presence of nicotine to address potential pharmacologic interactions on behavior. RO5126946 potentiated nicotine's effects on fear memory when both compounds were administered at subthreshold doses and did not interfere with procognitive effects observed when both compounds were administered at effective doses. Overall, RO5126946 is a novel α7nAChR PAM with cognitive-enhancing properties.


Subject(s)
Benzamides/pharmacology , Sulfonamides/pharmacology , alpha7 Nicotinic Acetylcholine Receptor/drug effects , Allosteric Regulation , Animals , Cells, Cultured , Cognition/drug effects , Hippocampus/drug effects , Humans , Learning/drug effects , Male , Memory/drug effects , Nicotine/pharmacology , Rats , Rats, Sprague-Dawley , Receptors, GABA-A/physiology , Receptors, Glutamate/physiology
6.
J Pharmacol Exp Ther ; 344(1): 23-32, 2013 Jan.
Article in English | MEDLINE | ID: mdl-23010360

ABSTRACT

Inhibition of cardiac late sodium current (late I(Na)) is a strategy to suppress arrhythmias and sodium-dependent calcium overload associated with myocardial ischemia and heart failure. Current inhibitors of late I(Na) are unselective and can be proarrhythmic. This study introduces GS967 (6-[4-(trifluoromethoxy)phenyl]-3-(trifluoromethyl)-[1,2,4]triazolo[4,3-a]pyridine), a potent and selective inhibitor of late I(Na), and demonstrates its effectiveness to suppress ventricular arrhythmias. The effects of GS967 on rabbit ventricular myocyte ion channel currents and action potentials were determined. Anti-arrhythmic actions of GS967 were characterized in ex vivo and in vivo rabbit models of reduced repolarization reserve and ischemia. GS967 inhibited Anemonia sulcata toxin II (ATX-II)-induced late I(Na) in ventricular myocytes and isolated hearts with IC(50) values of 0.13 and 0.21 µM, respectively. Reduction of peak I(Na) by GS967 was minimal at a holding potential of -120 mV but increased at -80 mV. GS967 did not prolong action potential duration or the QRS interval. GS967 prevented and reversed proarrhythmic effects (afterdepolarizations and torsades de pointes) of the late I(Na) enhancer ATX-II and the I(Kr) inhibitor E-4031 in isolated ventricular myocytes and hearts. GS967 significantly attenuated the proarrhythmic effects of methoxamine+clofilium and suppressed ischemia-induced arrhythmias. GS967 was more potent and effective to reduce late I(Na) and arrhythmias than either flecainide or ranolazine. Results of all studies and assays of binding and activity of GS967 at numerous receptors, transporters, and enzymes indicated that GS967 selectively inhibited late I(Na). In summary, GS967 selectively suppressed late I(Na) and prevented and/or reduced the incidence of experimentally induced arrhythmias in rabbit myocytes and hearts.


Subject(s)
Anti-Arrhythmia Agents/pharmacology , Arrhythmias, Cardiac/drug therapy , Cardiotonic Agents/pharmacology , Pyridines/pharmacology , Sodium Channel Blockers/pharmacology , Triazoles/pharmacology , Acetanilides/pharmacology , Action Potentials/drug effects , Animals , Arrhythmias, Cardiac/etiology , Arrhythmias, Cardiac/physiopathology , Cnidarian Venoms/pharmacology , Female , Flecainide/pharmacology , Heart Conduction System/drug effects , Long QT Syndrome/genetics , Long QT Syndrome/physiopathology , Mutation/physiology , Myocardial Ischemia/complications , Myocardial Ischemia/physiopathology , Myocytes, Cardiac/drug effects , Myocytes, Cardiac/metabolism , Patch-Clamp Techniques , Piperazines/pharmacology , Potassium Channel Blockers/pharmacology , Quaternary Ammonium Compounds/pharmacology , Rabbits , Ranolazine
7.
Am J Physiol Cell Physiol ; 301(3): C577-86, 2011 Sep.
Article in English | MEDLINE | ID: mdl-21677263

ABSTRACT

Late Na(+) current (I(NaL)) and Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) are both increased in the diseased heart. Recently, CaMKII was found to phosphorylate the Na(+) channel 1.5 (Na(v)1.5), resulting in enhanced I(NaL). Conversely, an increase of I(NaL) would be expected to cause elevation of intracellular Ca(2+) and activation of CaMKII. However, a relationship between enhancement of I(NaL) and activation of CaMKII has yet to be demonstrated. We investigated whether Na(+) influx via Na(v)1.5 leads to CaMKII activation and explored the functional significance of this pathway. In neonatal rat ventricular myocytes (NRVM), treatment with the I(NaL) activators anemone toxin II (ATX-II) or veratridine increased CaMKII autophosphorylation and increased phosphorylation of CaMKII substrates phospholamban and ryanodine receptor 2. Knockdown of Na(v)1.5 (but not Na(v)1.1 or Na(v)1.2) prevented ATX-II-induced CaMKII phosphorylation, providing evidence for a specific role of Na(v)1.5 in CaMKII activation. In support of this view, CaMKII activity was also increased in hearts of transgenic mice overexpressing a gain-of-function Na(v)1.5 mutant (N(1325)S). The effects of both ATX-II and the N(1325)S mutation were reversed by either I(NaL) inhibition (with ranolazine or tetrodotoxin) or CaMKII inhibition (with KN93 or autocamtide 2-related inhibitory peptide). Furthermore, ATX-II treatment also induced CaMKII-Na(v)1.5 coimmunoprecipitation. The same association between CaMKII and Na(v)1.5 was also found in N(1325)S mice, suggesting a direct protein-protein interaction. Pharmacological inhibitions of either CaMKII or I(NaL) also prevented ATX-II-induced cell death in NRVM and reduced the incidence of polymorphic ventricular tachycardia induced by ATX-II in rat perfused hearts. Taken together, these results suggest that a Na(v)1.5-dependent increase in Na(+) influx leads to activation of CaMKII, which in turn phosphorylates Na(v)1.5, further promoting Na(+) influx. Pharmacological inhibition of either CaMKII or Na(v)1.5 can ameliorate cardiac dysfunction caused by excessive Na(+) influx.


Subject(s)
Amino Acid Substitution/physiology , Calcium-Calmodulin-Dependent Protein Kinase Type 2/metabolism , Heart Ventricles/metabolism , Myocytes, Cardiac/metabolism , Sodium Channels/metabolism , Sodium/metabolism , Acetanilides/pharmacology , Acetanilides/therapeutic use , Animals , Animals, Newborn , Calcium/metabolism , Calcium Signaling/drug effects , Calcium Signaling/physiology , Calcium-Binding Proteins/metabolism , Calcium-Calmodulin-Dependent Protein Kinase Type 2/antagonists & inhibitors , Caspase 3/metabolism , Cell Death/drug effects , Cell Survival/drug effects , Cnidarian Venoms/pharmacology , Dose-Response Relationship, Drug , Electrophysiological Phenomena/drug effects , Electrophysiological Phenomena/physiology , Female , Gene Expression/drug effects , Heart Ventricles/cytology , Heart Ventricles/drug effects , Humans , Mice , Mice, Inbred Strains , Mice, Transgenic , Myocytes, Cardiac/drug effects , NAV1.5 Voltage-Gated Sodium Channel , Peptides/pharmacology , Peptides/therapeutic use , Perfusion , Phosphorylation/drug effects , Piperazines/pharmacology , Piperazines/therapeutic use , Protein Binding/drug effects , Protein Binding/physiology , RNA, Small Interfering/genetics , Rabbits , Ranolazine , Rats , Rats, Sprague-Dawley , Ryanodine Receptor Calcium Release Channel/metabolism , Sodium Channels/genetics , Sodium-Calcium Exchanger/antagonists & inhibitors , Sodium-Calcium Exchanger/metabolism , Tachycardia, Ventricular/chemically induced , Tachycardia, Ventricular/prevention & control , Tetrodotoxin/pharmacology , Veratridine/pharmacology
8.
Heart Rhythm ; 15(2): 277-286, 2018 02.
Article in English | MEDLINE | ID: mdl-29017927

ABSTRACT

BACKGROUND: Eleclazine (GS-6615) is a sodium channel blocker designed to improve the selectivity for cardiac late Na+ current (INa) over peak INa. OBJECTIVES: The goals of this study were to investigate the inhibition of late INa by eleclazine using a sample of long QT syndrome type 3 (LQT3) and overlap LQT3/Brugada syndrome mutant channels; to compare the apparent binding rates for eleclazine with those for other class 1 antiarrhythmic agents; and to investigate the binding site. METHODS: Wild-type human cardiac voltage-gated sodium channel (hNaV1.5) and 21 previously reported variants were studied using patch clamp recordings from a heterologous expression system. RESULTS: Eleclazine inhibited anemone toxin II-enhanced late INa from wild-type hNaV1.5 with a drug concentration that causes 50% block of 0.62 ± 0.12 µM (84-fold selectivity over peak INa). The drug concentration that causes 50% block of eleclazine to inhibit the enhanced late INa from LQT3 mutant channels ranged from 0.33 to 1.7 µM. At predicted therapeutic concentrations, eleclazine and ranolazine inhibited peak INa to a similar degree as assessed with 4 overlap LQT3/Brugada syndrome mutations. Eleclazine was found to interact with hNaV1.5 significantly faster than ranolazine and 6 other class 1 antiarrhythmic agents. Engineered mutations (F1760A/Y1767A) located within the local anesthetic binding site decreased the inhibition of late INa and peak INa by eleclazine. CONCLUSION: At predicted therapeutic concentrations, eleclazine elicits potent inhibition of late INa across a cohort of NaV1.5 mutant channels. These properties are consistent with a class 1b antiarrhythmic agent that associates with unusually rapid binding/unbinding rates.


Subject(s)
Cardiac Conduction System Disease/drug therapy , Long QT Syndrome/drug therapy , Myocytes, Cardiac/metabolism , Oxazepines/therapeutic use , Action Potentials , Cardiac Conduction System Disease/metabolism , Cardiac Conduction System Disease/physiopathology , Humans , Long QT Syndrome/metabolism , Long QT Syndrome/physiopathology , Myocytes, Cardiac/drug effects , Myocytes, Cardiac/pathology , Patch-Clamp Techniques , Sodium Channel Blockers/therapeutic use
9.
Neurochem Int ; 48(8): 703-7, 2006 Jun.
Article in English | MEDLINE | ID: mdl-16487630

ABSTRACT

Neurosteroids are modulators of several receptors and ion channels and are implicated in the pathophysiology of several neuropsychiatric diseases including hepatic encephalopathy (HE). The neurosteroid, allopregnanolone, a positive allosteric modulator of GABA(A) receptors, accumulates in the brains of HE patients where it can potentiate GABA(A) receptor-mediated responses. Attenuation of the effects of neurosteroids on GABA-ergic neurotransmission is therefore of interest for the management of HE. In the present study, we determined the effect of the benzodiazepine partial inverse agonist, Ro15-4513, and the benzodiazepine antagonist, flumazenil on modulation of the GABA(A) mediated chloride currents by allopregnanolone and on spontaneous synaptic activity in cultured hippocampal neurons using the patch-clamp technique. Allopregnanolone (0.03-0.3 microM), dose-dependently potentiated GABA-induced currents, an action significantly reduced by Ro15-4513 (10 microM). In contrast, flumazenil (10 microM) had no effect on the ability of allopregnanolone to potentiate GABA(A) currents but it blocked the effects of Ro15-4513. The frequency of spontaneous synaptic activity was significantly reduced in the presence of allopregnanolone (0.1 microM) from 1.5+/-0.7 to 0.1+/-0.04Hz. This action was partially reversed by Ro15-4513 (10 microM) but was not significantly influenced by flumazenil (10 microM). These findings suggest that the beneficial affects of Ro15-4513 in experimental HE result from attenuation of the effects of neurosteroids at GABA(A) receptors. Our results may provide a rational basis for the use of benzodiazepine inverse agonists in the management and treatment of hepatic encephalopathy in patients with liver failure.


Subject(s)
Benzodiazepines/pharmacology , Hippocampus/metabolism , Neurons/metabolism , Pregnanolone/pharmacology , Receptors, GABA-A/drug effects , Affinity Labels/pharmacology , Allosteric Regulation/drug effects , Allosteric Regulation/physiology , Animals , Azides/pharmacology , Cells, Cultured , Chloride Channels/drug effects , Chloride Channels/metabolism , Dose-Response Relationship, Drug , Drug Interactions/physiology , Female , Flumazenil/pharmacology , GABA Modulators/pharmacology , Hepatic Encephalopathy/drug therapy , Hepatic Encephalopathy/metabolism , Hepatic Encephalopathy/physiopathology , Hippocampus/cytology , Hippocampus/drug effects , Male , Neural Inhibition/drug effects , Neural Inhibition/physiology , Neurons/drug effects , Patch-Clamp Techniques , Pregnanolone/metabolism , Rats , Rats, Wistar , Receptors, GABA-A/metabolism , Synaptic Transmission/drug effects , Synaptic Transmission/physiology
10.
J Med Chem ; 59(19): 9005-9017, 2016 Oct 13.
Article in English | MEDLINE | ID: mdl-27690427

ABSTRACT

Late sodium current (late INa) is enhanced during ischemia by reactive oxygen species (ROS) modifying the Nav 1.5 channel, resulting in incomplete inactivation. Compound 4 (GS-6615, eleclazine) a novel, potent, and selective inhibitor of late INa, is currently in clinical development for treatment of long QT-3 syndrome (LQT-3), hypertrophic cardiomyopathy (HCM), and ventricular tachycardia-ventricular fibrillation (VT-VF). We will describe structure-activity relationship (SAR) leading to the discovery of 4 that is vastly improved from the first generation late INa inhibitor 1 (ranolazine). Compound 4 was 42 times more potent than 1 in reducing ischemic burden in vivo (S-T segment elevation, 15 min left anteriorior descending, LAD, occlusion in rabbits) with EC50 values of 190 and 8000 nM, respectively. Compound 4 represents a new class of potent late INa inhibitors that will be useful in delineating the role of inhibitors of this current in the treatment of patients.

11.
Neuropharmacology ; 62(7): 2251-60, 2012 Jun.
Article in English | MEDLINE | ID: mdl-22313527

ABSTRACT

Ranolazine, an anti-anginal drug, reduces neuropathic and inflammatory-induced allodynia in rats. However, the mechanism of ranolazin's anti-allodynic effect is not known. We hypothesized that ranolazine would reduce dorsal root ganglion (DRG) Na(+) current (I(Na)) and neuronal firing by stabilizing Na(+) channels in inactivated states to cause voltage- and frequency-dependent block. Therefore, we investigated the effects of ranolazine on tetrodotoxin-sensitive (TTXs) and tetrodotoxin-resistant (TTXr) I(Na) and action potential parameters of small diameter DRG neurons from embryonic rats. Ranolazine (10 and 30 µM) significantly reduced the firing frequency of evoked action potentials in DRG neurons from 19.2 ± 1.4 to 9.8 ± 2.7 (10 µM) and 5.7 ± 1.3 (30 µM) Hz at a resting membrane potential of -40 mV. Ranolazine blocked TTXs and TTXr in a voltage- and frequency-dependent manner. Furthermore, ranolazine (10 µM) blocked hNa(v)1.3 (expressed in HEK293 cells) and caused a hyperpolarizing shift in the voltage dependence of steady-state intermediate and slow inactivation Na(v)1.3 current. Taken together, the results suggest that ranolazine suppresses the hyperexcitability of DRG neurons by interacting with the inactivated states of Na(+) channels and these actions may contribute to its anti-allodynic effect in animal models of neuropathic pain.


Subject(s)
Acetanilides/pharmacology , Action Potentials/physiology , Ganglia, Spinal/embryology , Ganglia, Spinal/physiology , NAV1.3 Voltage-Gated Sodium Channel/physiology , Piperazines/pharmacology , Sodium Channel Blockers/pharmacology , Sodium Channels/physiology , Action Potentials/drug effects , Animals , Cells, Cultured , Ganglia, Spinal/drug effects , HEK293 Cells , Humans , Ranolazine , Rats
12.
Neurochem Int ; 59(3): 404-12, 2011 Sep.
Article in English | MEDLINE | ID: mdl-21315124

ABSTRACT

Human pluripotent stem cells have enormous potential value in neuropharmacology and drug discovery yet there is little data on the major classes and properties of receptors and ion channels expressed by neurons derived from these stem cells. Recent studies in this lab have therefore used conventional patch-clamp electrophysiology to investigate the pharmacological properties of the ligand and voltage-gated ion channels in neurons derived and maintained in vitro from the human stem cell (hSC) line, TERA2.cl.SP12. TERA2.cl.SP12 stem cells were differentiated with retinoic acid and used in electrophysiological experiments 28-50 days after beginning differentiation. HSC-derived neurons generated large whole cell currents with depolarizing voltage steps (-80 to 30 mV) comprised of an inward, rapidly inactivating component and a delayed, slowly deactivating outward component. The fast inward current was blocked by the sodium channel blocker tetrodotoxin (0.1 µM) and the outward currents were significantly reduced by tetraethylammonium ions (TEA, 5 mM) consistent with the presence of functional Na and K ion channels. Application of the inhibitory neurotransmitters, GABA (0.1-1000 µM) or glycine (0.1-1000 µM) evoked concentration dependent currents. The GABA currents were inhibited by the convulsants, picrotoxin (10 µM) and bicuculline (3 µM), potentiated by the NSAID mefenamic acid (10-100 µM), the general anaesthetic pentobarbital (100 µM), the neurosteroid allopregnanolone and the anxiolytics chlordiazepoxide (10 µM) and diazepam (10 µM) all consistent with the expression of GABA(A) receptors. Responses to glycine were reversibly blocked by strychnine (10 µM) consistent with glycine-gated chloride channels. The excitatory agonists, glutamate (1-1000 µM) and NMDA (1-1000 µM) activated concentration-dependent responses from hSC-derived neurons. Glutamate currents were inhibited by kynurenic acid (1 mM) and NMDA responses were blocked by MgCl(2) (2 mM) in a highly voltage-dependent manner. Together, these findings show that neurons derived from human stem cells develop an array of functional receptors and ion channels with a pharmacological profile in keeping with that described for native neurons. This study therefore provides support for the hypothesis that stem cells may provide a powerful source of human neurons for future neuropharmacological studies.


Subject(s)
Cell Differentiation , Neurons/drug effects , Stem Cells/cytology , Chlordiazepoxide/pharmacology , Diazepam/pharmacology , Glycine/pharmacology , Humans , Immunohistochemistry , Ion Channel Gating , Ligands , Mefenamic Acid/pharmacology , Neurons/cytology , Pentobarbital/pharmacology , Pregnanolone/pharmacology , Tetraethylammonium/pharmacology , Tetrodotoxin/pharmacology , gamma-Aminobutyric Acid/pharmacology , gamma-Aminobutyric Acid/physiology
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